HIV Associated Neurocognitive Disorder (HAND) is one of the most common and clinically important complications of HIV infection. Neurotoxicity is due to the accumulation of HIV-encoded Trans Activator of Transcription (Tat) and other viral proteins released from infected cells. Neuronal damage in HIV-infected patients is significantly exacerbated by drug abuse. Thus, there is an urgent and increasing need for effective therapeutic strategies for HAND. We utilized a computational approach in our NIH-funded work to uncover DEAD Box RNA Helicase 3 (DDX3) as a potential target in HAND. DDX3 was recently established as a target protein for cancer therapy. A selective small molecule inhibitor of DDX3 helicase activity, RK-33, has recently been developed and tested in animal models of lung cancer. However, DDX3 has never been proposed to be involved in HAND development, and RK-33 has never been tested in models of HAND or other neurodegenerative diseases. We therefore analyzed the effects of the DDX3 inhibitor RK-33 on the viability of Tat/cocaine-treated primary mouse cortical neurons. Our preliminary results show that RK-33 protects these neurons against the damage caused by a combined insult of Tat and cocaine, and that the effect was quite robust. Thus, the hypothesis of this proposal is that the activity of DDX3 promotes drug-induced HAND-associated neuropathology via pathological Stress Granules (SGs), and the inhibition of DDX3 enzymatic activity alleviates this neurotoxicity by interfering with pathological SG formation. The main goal of the proposal is to establish DDX3 as a target for anti-HAND drug development, validate that pharmacological inhibition of DDX33, can be a treatment for HAND, and also uncover important mechanistic links in HAND neuropathology. Aim 1 will determine the mechanism of RK-33 protection of cortical neurons from the combined neurotoxicity of Tat and cocaine. Published evidence suggests that Tat interacts with DDX3, and that this interaction depends on the enzymatic activity of DDX3. We propose that Tat interacts with DDX3 to facilitate Tat/cocaine compounded neurotoxicity, and the protective effects of RK-33 result from inhibition of DDX3?s enzymatic activity. Aim 2 will establish the function of SGs as a central regulator of the protective effects of RK-33 inhibition against Tat /cocaine-induced neurotoxicity. DDX3 is a core component of SGs and is believed to directly participate in their assembly. The accumulation of pathological SGs is a noticeable feature of amyotrophic lateral sclerosis and other neurodegenerative diseases. We propose that DDX3 inhibition by RK-33 protects the neurons from Tat/cocaine by impairing pathological SG assembly and dynamics. Over the course of this project, the role of DDX3 in the pathology of HAND will be better understood. Determining a connection of DDX3 to pathology associated with SGs could have much broader implications for many other neurodegenerative diseases. Going forward, pharmacological DDX3 inhibition will be validated in vivo, pursued with drug development efforts, and could ultimately lead to new clinical treatment options for otherwise untreatable neurodegenerative diseases.